Abstract
The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K(m) value for d-lactate of approximately 160 microm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K(m) value of approximately 580 microm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in beta-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.
Highlights
L-lactate dehydrogenase belongs to the most intensely studied enzyme families [2, 3], our knowledge about the structure, kinetics, and biological function of D-LDH3 is limited
It is assumed that the role of the MG pathway, from MG synthase to D-lactate cytochrome c oxidoreductase in the extant metabolism, is to detoxify MG, whereas in the early state of metabolic development it might function as an anaplerotic route for the tricarboxylic acid cycle [15]
In Silico Identification of D-LDH Orthologous Proteins in A. thaliana and Phylogenetic Analysis of Homologs—To identify candidate genes encoding D-LDH enzymes, the A. thaliana genome was searched with the S. cerevisiae D-LDH (ScD-LDH or DLD1, UniProtKB/Swiss-Prot accession number P32891) protein sequence, using the BLAST search algorithm
Summary
D-LDH, D-lactate dehydrogenase; D-2HG, D-2-hydroxyglutarate; D-2HGDH, D-2-hydroxyglutarate dehydrogenase; DCIP, dichlorophenolindophenol; D-LDH, D-lactate dehydrogenase; ETF, electron transfer protein; ETFQO, ETF-ubiquinone oxidoreductase; MG, methylglyoxal; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Ni2ϩ-NTA, Ni2ϩ-nitrilotriacetic acid; PMS, phenazine methosulfate; DCIP, 2,6-dichlorophenolindophenol; MS, Murashige and Skoog; D-2HB, D-2-hydroxybutyrate. Glyoxalase I and II activities are present in all tissues of eukaryotic organisms. Atlante et al [13] showed that externally added D-lactate caused oxygen consumption by mitochondria and that this metabolite was oxidized by a mitochondrial flavoprotein in Helianthus tuberosus. The isolation and structural and biochemical characterization of the recombinant mature D-LDH from A. thaliana (AtD-LDH) and its paralog, which was found to be a D-2-hydroxyglutarate dehydrogenase (AtD-2HGDH), is described. Whereas AtD-LDH has a narrow substrate specificity and the preferred substrates are D-lactate and D-2-hydroxybutyrate, AtD-2HGDH showed activity exclusively with D-2-hydroxyglutarate. Based on gene coexpression analysis and analysis of corresponding knock-out mutants, the participation of these previously unrecognized mitochondrial activities in plant metabolism is discussed
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